Receptor-ligand interactions are critical components of many fundamental biological processes. Such interactions involve specific binding of a macromolecule receptor (e.g., enzyme, cell-surface protein, antibody or oligonucleotide) to a particular ligand molecule. Receptor-ligand binding may affect any of a variety of intercellular and intracellular processes in an organism, such as signal transduction, gene expression, immune responses or cell adhesion. An improved understanding of receptor-ligand interactions is necessary for many areas of research in the life sciences, as well as for the development of agents that modulate such interactions for therapeutic and other applications.
Miniaturized ligand-arrays, formed using microfabrication and solid-phase chemical synthesis on substantially planar supports, have been used to facilitate the study of receptor-ligand interactions (for representative examples, see Fodor et al., Science (1991) 251:767; Pease et al., Proc. Natl. Acad. Sci. USA 91:5022, 1994; Pirrung et al., U.S. Pat. No. 5,405,783; Fodor et al., U.S. Pat. No. 5,445,934; Pirrung et al., U.S. Pat. No. 5,143,854; Fodor et al., U.S. Pat. No. 5,424,186 and Fodor et al., U.S. Pat. No. 5,510,270; Chee et al., Science (1996) 274:610 and Brennan, U.S. Pat. No. 5,474,796). Contacting a ligand array with labeled receptor allows many ligands to be simultaneously screened for receptor binding. The location of bound receptor on the array is determined by detecting photons or radioactivity. However, the surface density of ligand is often low, resulting in the need for costly imaging equipment and long image acquisition times. Drug discovery efforts have been further hampered by low ligand surface density, since many functional assays require higher ligand concentrations to identify drug leads.
One approach to increasing surface density of ligands involves immobilizing ligands on an array of polyacrylamide pads using microfabrication techniques (see Guschin et al., Anal. Biochem. 250:203, 1997 and Yershov et al., Proc. Natl. Acad. Sci. USA 93:4913, 1996). Such an approach increases the surface density of the ligands, but places a size restriction on diffusion into the polymer that many receptors exceed. Furthermore, such polymeric supports may not be compatible with solid-phase chemical synthesis, which requires adequate swelling and salvation of a polymeric matrix in order to achieve efficient mass transfer of reagents. Further, although this polymer can be photopatterned (i.e., multiple discrete pads may bc generated by a process involving exposure to irradiation), the photosensitivity is severely limited, requiring 30 minutes of illumination. Such a low throughput is inadequate for mass production.
Existing techniques for increasing ligand density on a solid support provide insufficient surface area enhancement. Such techniques include the use of acid-etched porous silicon and an electrochemically manufactured metal oxide membrane as substrates for detecting the specific binding of ligands by receptors (see Beattie et al., Clin. Chem. 41:700, 1995 and Van Damme and Kreuwel, WO99/02266). The porous silicon is macroporous with 3 to 5 micron diameter pores arranged in parallel and oriented perpendicular to the substrate surface. Relative to nanoporous materials, a macroporous configuration has inadequate surface area to significantly increase ligand surface density. Although the electrochemically manufactured metal oxide membrane has pores as small as 0.2 microns, it too provides little surface area enhancement with only a 10-fold increase in surface area for each micron of membrane thickness.
Additionally, the parallel pore orientation of these substrates is technically cumbersome, since it requires a flow-through apparatus in order for receptor to bind ligand. Further, it is unclear whether such substrates could function as solid supports for multiple rounds of synthetic reactions. The electrochemically manufactured metal oxide membrane also suffers from incompatibility with microfabrication methods.
Rigid porous supports that do not require swelling in solvents and are compatible with attachment of ligands or receptors offer the potential to increase ligand surface density by providing a high surface area for ligand attachment. For example, porous bodies have been made from slurries consisting of a binder and particles having a high surface area (see Messing, U.S. Pat. No. 3,910,851 and Messing, in: Methods in Enzymology, vol. XLIV, p. 149, edited by Klaus Mosbach, (1976), Academic Press N.Y.). However, to date, porous supports and coatings have not been successfully applied to microfabrication of ligand arrays.
Porous coatings with controlled porosity have been obtained by sol-gel and particulate methods (see Frye et al., U.S. Pat. No. 5,224,972; Frye et al., U.S. Pat. No. 5,589,396; Suppiah, U.S. Pat. No. 5,120,600 and Frye et al., in: Better Ceramics Through Chemistry IV, vol. 180, Mat. Res. Soc. Symp. Proc., edited by Brinker et al., (1990), p. 583). Such methods produce controlled porous coatings with chemically modified surfaces for the purpose of providing steric and chemical selectivity to a sensor surface, via nonspecific molecular interactions (e.g. chelation and ion exchange). Such coatings have not been used as supports for detecting the specific binding characteristic of macromolecular receptors or to create arrays of complex ligands. Further such coatings cannot be made greater than one micron thick without multiple coats, and have not been successfully patterned by microfabrication methods.
Other porous coatings suffer from incompatibility with solid phase ligand synthesis. From the field of imaging, positive and negative images can be formed in coatings of photosensitized colloidal particles (see Pu et al., J Imaging Sci. 33:177, 1989). Such coatings consist of a phenolic resin (0% to 15%), a bis-azide (optional), and colloidal particles encapsulated by organic polymer, diacid chlorides, and photoactive azide groups. Although these coatings may be patterned using microfabrication techniques, they have not been used to increase ligand surface density, detect ligand-receptor binding or prepare ligand arrays by solid-phase chemical synthesis. In fact, organic solvents would be expected to swell and distort existing coatings, making them incompatible with solid-phase synthesis.
Still further porous inorganic coatings have been designed to reduce reflectivity. For example, both aged and unaged colloidal dispersions have been used to form continuous porous coatings of uniform thickness (see Cathro et al., Solar Energy 32:573, 1984 and Lange et al., U.S. Pat. No. 4,816,333). The resulting dried coatings are from about 0.02 μm to 0.50 μm thick. Although the surface area of a porous coating may be increased by increasing its thickness, uniform colloidal coatings greater than about 1.5 μm thick cannot be obtained without using certain additives (see Daniels et al., in: Better Ceramics Through Chemistry VII, vol. 435, Mat. Res. Soc. Symp. Proc., edited by Coltrain et al., (1996), p. 215). Even with additives, such coatings still form cracks. In fact, colloidal coatings typically are nonuniform and discontinuous (see Moulton, U.S. Pat. No. 2,601,123). Further, colloidal coatings have not been patterned using microfabrication techniques, or used to increase ligand surface density, detect ligand-receptor binding, or prepare ligand arrays by solid-phase chemical synthesis.
Accordingly, there is a need in the art for methods for increasing ligand density on a surface in a manner that is fully compatible with microfabrication. In particular, there is a need for improved articles for use in the detection of macromolecular receptor binding, and the production of ligand arrays by solid-phase synthetic methods. The present invention fulfills these needs and further provides other related advantages.